Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
  • G01N33/728—Bilirubin; including biliverdin
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/814—Enzyme separation or purification
  • Y10S435/816—Enzyme separation or purification by solubility
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/911—Microorganisms using fungi

Definitions

• This invention relates to fungal enzyme compositions having bilirubin activity, their use and their preparation.
• Bilirubin is a yellow substance which is formed in the blood by degradation of haemoglobin.
• the rapid and accurate detection of bilirubin in blood serum is vitally important to medical diagnosis of disease states, e.g., jaundice, in human beings.
• H. Plieninger and L. Petzold, in Z. Physiol. Chem., 297:238, 1954 describe what they refer to as "mushroom enzymes" having activity on bilirubin.
• the enzymatic activity is reportedly obtained merely by incubating mushroom juice from a mushroom described as Psalliota campestris (believed to be either Agaricus bisporus or Agaricus campestris) with bilirubin.
• Psalliota campestris believed to be either Agaricus bisporus or Agaricus campestris
• Plieninger et al base their findings solely on observation of oxygen uptake when a bilirubin medium was incubated with mushroom juice. They reported no oxygen uptake upon incubation of the bilirubin medium by itself.
• reaction time scale reported by Plieninger et al in terms of hours is highly atypical of enzyme catalyzed reactions, which characteristically occur within minutes or less.
• comparative data in Example 1 . infra show, we have found no evidence that simple mushroom juice exhibits enzymatic activity on bilirubin.
• the present invention provides a bilirubin-specific enzyme composition characterized in that it comprises a fungal enzyme having a protein content which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37°C, degrades at least about 0.02 micromole of bilirubin per minute per milligram of protein.
• the fungal enzyme composition of the invention may be extracted from mushrooms, preferably from the genus Agaricus, more preferably the species Agaricus bisporus.
• An especially preferred embodiment of the invention provides a fungal enzyme composition which not only degrades bilirubin but also generates detectable amounts of hydrogen peroxide.
• the bilirubin-specific hydrogen peroxide-generating fungal enzyme composition is employed with a hydrogen peroxide detection composition to provide an assay for bilirubin especially bilirubin in an aqueous liquid.
• the fungal enzyme composition may also be used to remove bilirubin as an interferent from aqueous samples to be assayed for analytes other than bilirubin.
• compositions are their specific activity on bilirubin.
• biliverdin or haemoglobin both of which are highly coloured and chemically similar to bilirubin, no visible change occurs. This indicates inactivity on substances closely related to bilirubin.
• composition obtained by extraction Method II described below, has especially high bilirubin activity and, in addition, will generate hydrogen peroxide.
• the preferred fungal enzyme composition of the invention by degrading bilirubin, yields a reaction product exhibiting characteristic absorption and emission spectra.
• a reaction product When the fungal enzyme composition is incubated with a bilirubin-containing aqueous liquid at about 37°C and about 7.4 pH in a 0.05 M sodium phosphate buffer, the reaction product exhibits an absorption peak at about 510 nm and, upon excitation with 450 nm wavelength radiation, fluoresces at about 525 nm.
• reaction products have not been fully characterized, however, it is not known whether the substance responsible for the absorption and fluorescence characteristics is a single compound or a mixture. It may well be that the particular compound in the reaction product which exhibits an absorption peak at 510 nm is different from that which fluoresces at 525 nm. Nevertheless, because neither bilirubin nor the enzyme preparation alone exhibits these absorption and fluorescence characteristics, it is evident that the characteristics belong to one or more compounds in the reaction product.
• Especially preferred fungal enzyme compositions of the invention not only degrade bilirubin, but also generate hydrogen peroxide.
• Such a fungal enzyme composition is particularly suited as an assay composition for bilirubin because it can readily be coupled with known hydrogen peroxide detection compositions, e.g., enzymatic hydrogen peroxide detectors.
• the latter preferably contain a material having peroxidative activity, particularly peroxidase, and an indicator, e.g., a chromogenic indicator.
• chromogenic indicator includes (a) substances which change colour in the presence of hydrogen peroxide and a material having peroxidative activity and (b) substances, preferably mixtures of substances, which undergo no substantial colour change upon oxidation in the presence of hydrogen peroxide and a material having peroxidative activity, but which substances, upon oxidation, react with a colour-forming or coloui-changing substance (e.g., a coupler) to give visible evidence of chemical reaction.
• a colour-forming or coloui-changing substance e.g., a coupler
• chromogenic indicators In addition to chromogenic indicators, other indicators may also be employed.
• a bilirubin-specific, hydrogen peroxide-generating fungal enzyme composition and a material having peroxidative activity can be incorporated into a membrane of an oxygen-sensitive polarographic electrode of the kind described in Rawls, Rebecca L., "Electrodes Hold Promise In Biomedical Uses", Chemical and Engineering News, January 5, 1976, page 19.
• the oxygen sensitive polarographic electrode serves as the indicator.
• the present enzyme and assay compositions may be employed to detect or determine bilirubin in an aqueous liquid sample by:
• the assay composition comprises a hydrogen peroxide-generating fungal enzyme composition of the invention and a hydrogen peroxide detection composition thereby providing a completely enzymatic assay for bilirubin.
• the above-described assay composition and method are particularly effective at a pH of about 7.4 and a temperature from about 20°C to about 40°C, they can be used over wider pH and temperature ranges.
• the fungal enzyme compositions of the invention provide useful bilirubin-degrading activity over a pH range from about 7.3 to about 8.0 and a temperature range from about 20°C to about 50°C.
• a buffer is also present in the assay composition to maintain the pH during the assay within the effective pH range of the fungal enzyme composition.
• Phosphates such as sodium phosphate are particularly suitable.
• other buffers are appropriate and are described, for example, by Good in Biochemistry, 5, 467 (1966).
• the amounts of the various components of the assay composition may vary widely. Depending upon the range of bilirubin concentrations for which the composition is intended, one uses more or less of the bilirubin-specific enzyme preparation. When using a fungal enzyme composition obtained by extraction Method II to analyze for a bilirubin concentration varying from about 0.1 to about 10 milligrams per decilitre, one would usually employ an assay composition containing an enzyme composition having the minimum activity specified above in an amount of about 0.1 to about 0.3 mg. When using a more preferred fungal enzyme composition of the invention having an activity level from about 2 to 5 or more times higher than the specified minimum, proportionately smaller amounts will, of course, be used.
• the amounts of optional hydrogen peroxide detection composition and optional buffer may vary widely.
• the amount of hydrogen peroxide detection composition will also depend on the bilirubin concentration for which the assay composition is intended, as well as on the purity and activity of the fungal enzyme composition in the assay composition.
• a further embodiment of the invention provides a method for the assay or detection of an analyte other than bilirubin in an aqueous liquid sample containing bilirubin, bilirubin being an interferent for said assay method, said method comprising
• interactive composition may be any composition capsule of physical, electrical, chemical or other interaction with the analyte of interest leading to a detestable change which can be related to the presence and/or amount of the analyte.
• this interactive composition may be a single compound, the term is employed broadly herein to include multi-component compositions.
• the term includes a multi-component composition wherein a first component reacts with the analyte, and the reaction product of such reaction then reacts with a second component to produce a further reaction product which exhibits the desired detectable change.
• a fungal enzyme composition of the invention When a fungal enzyme composition of the invention is employed to eliminate or reduce bilirubin as an interferent in an assay, the interactive composition which is employed must itself be non- interfering with respect to the bilirubin-specific fungal enzyme composition. For example, if the analyte is to be detected by use of an interactive composition containing a hydrogen peroxide detection composition, it would clearly be inappropriate to use a fungal enzyme composition which itself generates hydrogen peroxide. Because the fungal enzyme composition of the invention can be produced to degrade bilirubin, either. with or without generation of hydrogen peroxide, this particular problem can readily be avoided.
• the detectable change produced in either of the two assay methods described above may be detected by a wide variety of means.
• a preferred embodiment of the invention employs a radiometric device capable of detecting electromagnetic energy, such as a colour change, or a change in fluorescent or radioactive emission.
• the product of interaction of the fungal enzyme composition of the invention with bilirubin exhibits fluorescence at about 525 nm as well as an absorption peak at 510 nm
• a spectrophotometer to measure the decrease in the characteristic absorption peak of bilirubin at 445-460 nm due to the degradation of bilirubin by the fungal enzyme composition.
• the assay composition and methods of the invention intended either for bilirubin assay or for removal of bilirubin as an interferent can be employed in liquid analytical techniques. These are sometimes called “wet chemistry”.
• the assay composition and methods can also be employed in analytical techniques employing dry test elements, sometimes called “dry chemistry”.
• dry chemistry techniques, the assay is carried out entirely in a liquid medium; and the fungal enzyme composition or the assay composition containing it is employed as a liquid reagent. In such case, it is preferred to employ the fungal enzyme composition or assay composition in admixture with aqueous liquid at a temperature of from about 20°C to about 40°C and at a pH from about 7.3 to about 8.0.
• the fungal enzyme composition and assay composition are employed in "dry chemistry" techniques, they can be incorporated, for example, by imbibition, impregnation, or by coating techniques, into a reagent zone of a dry test element, e.g., a reagent layer of a dip-and-read fibrous test strip or a reagent layer of a non-fibrous multilayer element as described in U.S. Patent 3,992,158 or U.K. Specification 1,440,464.
• a dry test element e.g., a reagent layer of a dip-and-read fibrous test strip or a reagent layer of a non-fibrous multilayer element as described in U.S. Patent 3,992,158 or U.K. Specification 1,440,464.
• the present invention provides a dry test element for the assay of an aqueous liquid which element contains a reaction zone characterized in that the reaction zone contains an enzyme composition according to the present invention or an assay composition according to the present invention.
• the fungal enzyme composition or assay composition is present as a dried residue, for example, as a lyophilized (i.e., freeze-dried) composition.
• the fungal enzyme composition and assay composition may be prepared and used in aqueous liquid form or as a dried residue, e.g., as a freeze-dried powder.
• the dried residue may be packaged and stored and later reconstituted with water immediately prior to use.
• Example 1 Control Example - Measurement of 0 2 Uptake of Bilirubin-Containing Medium in Presence and Absence of Mushroom Juice; A Repeat of Work Done by Plieninger et al, supra.
• This Example describes a fungal enzyme composition of the present invention prepared by Extraction Method I. It describes also a series of i tests of the fungal enzyme composition and shows the effect of variables.
• Haemoglobin - obtained from freshly isolated human whole blood or purchased from Sigma Chemical Co., St. Louis, Missouri.
• Biliverdin - purchased from Sigma Chemical Co.
• bilirubin is a degradation product of haemoglobin and bilirubin is easily oxidized to biliverdin, it was important to ascertain whether the fungal enzyme composition prepared according to the procedure described in Part A of this Example would react with either of these compounds.
• haemoglobin 10-15 mg/dl
• biliverdin 1-5 mg/dl
• the substrates were each incubated in 0.05 M phosphate buffer, pH 7.4, 22°C with a series of varying amounts of the enzyme preparation as described in Part A containing from 0.3 to 0.8 mg of protein.
• the assay reaction mixtures were scanned against reference cuvettes containing identical compositions, except that the enzyme preparation was omitted.
• haemoglobin there was no change in absorbance at 420, 540, 580 or 620 nm (known ⁇ max for haemoglobin).
• V i i.e., change in absorbance at 460 nm per minute
• 5 minute assays measured as total change in absorbance at 460 nm over 5 minutes
• the response was linear up to the addition of about 15 ⁇ l of fungal enzyme composition (containing 0.18 mg of protein) for the initial velocity (V i ), and up to the addition of about 10 ⁇ l of fungal enzyme composition (containing about 0.12 mg of protein) for the 5 minute assay.
• a series of constant volume 10 ⁇ l samples of the fungal enzyme composition of Part A (each containing approximately 0.12 mg protein) was prepared. Each 10 ⁇ l sample was then added to a 2 ml sample of aqueous liquid containing a different concentration of bilirubin. The effect of varying levels of bilirubin concentration on the fungal enzyme composition was then evaluated as described in "Procedures.”
• V i . and bilirubin concentration The relationship between V i . and bilirubin concentration is shown in Table I. An increase in V i with bilirubin concentration was evident until about 4 mg/dl bilirubin concentration, which was at the upper limit of detection of the spectrophotometer. Based on a Lineweaver-Burk transformation of the available data [see Lineweaver, H. and Burk, D., Journal of American Chemical Society, Vol. 56, p. 658 (1934)], the apparent extrapolated Michaelis constant, K m , of the enzyme for bilirubin corresponds to about 7.04 mg/dl.
• the bilirubin concentration for each assay reaction mixture evaluated in this Part was constant at 2 mg/dl.
• a series of enzyme mixtures was prepared, each containing a fungal enzyme composition as described in Part A and a different amount of 0.05 M phosphate buffer to provide a different pH level. All other assay conditions were as described in "Procedures.”
• Each enzyme mixture was then evaluated in a 2 ml assay reaction mixture by adding a 10 ⁇ l sample of each enzyme mixture to a bilirubin-containing aqueous liquid as described in “Procedures.”
• Table II shows the pH-activity profile. The optimum pH was 7.4. The sharp drop in activity at lower pH indicated either that the enzyme was more stable to alkali than to acid or that the bilirubin was more soluble, and hence more available to the enzyme, at an alkaline pH, or both.
• This Example describes fungal enzyme compositions of the invention as prepared by Extraction Method II.
• the thus suspended enzyme material was precipitated with ice-chilled acetone (1:1 v/v); and this precipitate was resuspended in 2 volumes of 0.05 M sodium phosphate buffer and reprecipitated with chilled acetone. The final resuspension and reprecipitation steps were repeated 2 times to give an additional 2-fold purification of the fungal enzyme composition. The final supernatant was discarded. The remaining brownish precipitate was a bilirubin-degrading, hydrogen'peroxide-generating fungal enzyme composition of the invention and was found to contain approximately 240 to 300 mg protein. This fungal enzyme composition was then freeze dried until ready for use.
• Varying levels of the fungal enzyme composition extracted as described in Part A of this Example were added to solutions containing 2 mg/dl of freshly prepared bilirubin in 0.05 M sodium phosphate buffer, pH 7.4 + 0.05 at 22-25°C and an assay was conducted as described in Procedure 2. Plotting the data in Table V below shows a nearly linear relationship between the initial velocity, V il ( ⁇ 44 Onm/min ) and the amount of enzyme protein used per assay.
• Test conditions were the same as in Part D of this Example, except that 10 ⁇ l samples (about 0.6 mg protein) of the fungal enzyme composition were added to varying amounts of bilirubin solution (0-5 mg/dl) in each sample. Owing to the high absorbance of bilirubin solutions, kinetics at concentrations above 3 mg/dl were not obtainable. The data thus obtained illustrated a linear relationship between the initial velocity of bilirubin degradation by the fungal enzyme composition and the bilirubin concentration.
• Varying concentrations of bilirubin (0-5 mg/dl) were added to a series of assay reaction mixtures containing: 50 ⁇ l of a 1% fresh solution of o-phenylenediamine in 0.05 M sodium phosphate, pH 7.45 + 0.05, 10 ⁇ l of peroxidase solution prepared by Procedure 1 of this Example, and an amount of fungal enzyme composition obtained as in Part A of this Example and containing about 0.78 mg of protein. (A control reaction mixture contained everything except the fungal enzyme composition.) Each assay reaction mixture was incubated at 25°C for 2-3 minutes and the reaction was followed photometrically by measuring the increase in absorbance of the oxidized o-phenylenediamine dye at 550 nm.
• Table VII depicts the strikingly linear relationship between the initial velocity, V i ( ⁇ A 440nm/min ) and bilirubin concentrations, i.e., V I increases with increasing bilirubin levels.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Molecular Biology (AREA)
• Wood Science & Technology (AREA)
• General Health & Medical Sciences (AREA)
• Hematology (AREA)
• Biotechnology (AREA)
• Immunology (AREA)
• Microbiology (AREA)
• Biomedical Technology (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Analytical Chemistry (AREA)
• Urology & Nephrology (AREA)
• Physics & Mathematics (AREA)
• Medicinal Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• General Engineering & Computer Science (AREA)
• Cell Biology (AREA)
• Biophysics (AREA)
• Food Science & Technology (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Enzymes And Modification Thereof (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Applications Claiming Priority (2)

Publications (3)

Family

ID=25424407

Family Applications (1)

Country Status (5)

Cited By (12)

Families Citing this family (9)

• 1978
  • 1978-05-19 US US05/907,640 patent/US4211844A/en not_active Expired - Lifetime
  • 1978-10-31 CA CA315,309A patent/CA1112588A/fr not_active Expired
• 1979
  • 1979-05-17 JP JP54059798A patent/JPS5811194B2/ja not_active Expired
  • 1979-05-18 EP EP79300881A patent/EP0005637B1/fr not_active Expired
  • 1979-05-18 DE DE7979300881T patent/DE2963334D1/de not_active Expired

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events